Structural insight into Vibrio cholerae EIIC sugar transporter dimer captured in a substrate-free inward-facing state

Abstract

The phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) is a central pathway for carbohydrate transport in bacteria and plays a critical role in nutrient acquisition, metabolism, and virulence. In Vibrio cholerae, the glucose-specific EIIC transporter is a key component of the PTS system, mediating the transport of sugars into the bacterial cell, coupled with phosphorylation during translocation. Here, we present the 3.68 Å cryo-electron microscopy (cryo-EM) structure of the dimeric EIIC transporter from Vibrio cholerae in its inward-facing, substrate-free conformation. The structure reveals a detailed arrangement of the scaffold and transport domains, stabilized by extensive inter- and intraprotomer interactions. Comparative analysis with substrate-bound inward-facing structures of EIIC from E. coli highlights conformational changes, providing insights into substrate release and the structural transitions required for alternating access. Notably, the observed substrate-free inward-facing conformation features a larger substrate-binding pocket, which is consistent with a state poised for glucose release into the cytoplasm. The formation of a unique intraprotomer disulfide bond between residues C240 and C254 stabilizes the interface between the scaffold and transport domains, potentially regulating transporter dynamics. These findings elucidate the structural basis for substrate release in the PTS system and underscore the dynamic nature of EIIC-mediated sugar transport. Our study enhances the understanding of PTS system function in Vibrio cholerae and highlights the EIIC transporter as a promising target for antimicrobial drug development. Disruption of sugar transport in this essential pathway could impair bacterial growth and virulence, suggesting a novel therapeutic strategy against cholera. These results provide a foundation for future investigations into the structural and functional dynamics of bacterial sugar transporters.

1. Introduction

The phosphoenolpyruvate-dependent sugar phosphotransferase system (PEP-PTS) is a fundamental mechanism by which bacteria acquire and metabolize sugars. This system utilizes phosphoenolpyruvate (PEP) to transfer a phosphate group to sugars during their transport across the bacterial membrane [1–3]. The PTS system is not only for nutrient acquisition and metabolism but also for bacterial virulence and adaptability to changing environmental conditions [4–12]. In Vibrio cholerae, the causative agent of cholera, the PTS system is indispensable for its ability to colonize diverse niches, including the human intestine. Efficient acquisition and metabolism of sugars such as glucose, mannose, and N-acetylglucosamine (NAG) are vital for bacterial survival, energy production, and virulence factor expression [13–17].

The PTS system comprises three main components: enzyme I (EI), heat-stable phosphocarrier protein (HPr), and enzyme II (EII). EI and HPr function as sugar-nonspecific energy-coupling proteins, whereas EII facilitates substrate-specific transport. The EII complex consists of EIIA, EIIB, and the membrane-integral EIIC, which mediate sugar transport via conformationally driven alternating access [3,18]. Structural insights into EIIC are essential for understanding substrate recognition, transport specificity, and conformational transitions during sugar translocation. The inward-facing conformation, in which the transporter is poised to release sugar molecules into the cytoplasm after translocation, represents a crucial stage in the transport cycle. High-resolution structural data of this conformation can illuminate the molecular mechanisms underpinning substrate release and transporter dynamics [19].

Inhibiting the EIIC sugar transporter in Vibrio cholerae impairs key systems that the bacterium uses to sense and respond to nutrient availability, leading to several downstream effects that reduce its pathogenic potential. Specifically, blocking EIIC impairs sugar uptake and metabolism, disrupting central metabolic pathways essential for energy production and growth in nutrient-variable environments such as the intestine [20]. Furthermore, the loss of EIIC function significantly reduces biofilm formation by impairing necessary signaling pathways, thereby affecting environmental persistence and early host colonization stages [21]. Additionally, disrupted EIIC-mediated sugar transport interferes with nutrient-sensing circuits regulating key virulence factors, including cholera toxin, undermining the ability of bacteria to establish infection [22]. Thus, inhibiting EIIC in Vibrio cholerae profoundly affects sugar uptake, biofilm formation, and virulence gene activation, collectively undermining bacterial survival and pathogenicity.